Influenza viruses are major respiratory tract pathogens that present significant risks to individuals of all ages, particularly during pandemics. These risks would be greatly amplified should the lethal avian influenza strains, such as H5N1 and H7N9, become capable of human-to-human transmission, or if a new recombinant strain were engineered as a bioterror agent. Although vaccination can prevent influenza-associated morbidity and mortality, it does not do so in some people, even in healthy adults. This proposal seeks to leverage technological advances in the Robinson and Blish laboratories to build on findings from prior CCHI studies at Stanford that highlighted environmental exposures and the diversity and maturity of the lymphocyte repertoire as critical factors influencing vaccine responses. We hypothesize that prior environmental exposures influence the maturity and diversity of the immune repertoire and responses to different vaccines and play a greater role than genetics in generating effective vaccine-induced immunity against influenza. To investigate this hypothesis, we will use technologies recently developed in the Robinson and Blish laboratories to comprehensively define the phenotypic and functional repertoires of B cells, T cells and NK cells responding to influenza vaccination. We will couple our unique ability to measure the diversity, clonality, and functions of T, B, and NK cell populations at the single-cell level with the resources, expertise, and emerging technologies provided by the other U19 Projects and Cores (e.g., blood samples and clinical data from an extensive twin cohort assembled by the Clinical Core; CyTOF provided by the HIMC and developed by Project 4). In Aim 1, we will evaluate B cell, T cell and NK cell responses to influenza vaccination in monozygotic and dizygotic twins to determine the role of genetics and environment in the response to vaccination, comparing concordance in monozygotic twin pairs to that in dizygotic twin pairs. In Aim 2, we will determine how the vaccination method influences lymphocyte diversity and maturity, by comparing the B, T, and NK cell repertoire and responsiveness between monozygotic twin pairs receiving intranasally administered live attenuated vaccine (LAIV) and those receiving parenterally administered inactivated flu vaccine (TIV). In Aim 3, we will perform an integrated analysis of the datasets from this and other U19 Projects and Cores to determine the optimal levels of immune diversity and maturity that predict effective vaccination. Thus, by dissecting an extensive twin cohort with state-of-the-art tools, we propose to deliver a singularly detailed, integrated picture of the mechanisms governing human immune responses to influenza vaccination.